For in vitro diagnostic use. Others cobas EZH2 Mutation Test IVD cobas® EZH2 Mutation Test RMD-4800-EZH2-001 07 258 321 190 7 258 321 190 07258321190 7258321190 07258321190 KIT COBAS 4800 EZH2 AMP/DET 24T IVD cobas EZH2 Mutation Test 00875197005592 Reagents, kits 1 kit 24 tests true The cobas® EZH2 Mutation Test is a real-time allele-specific PCR test for qualitative detection of single nucleotide mutations for Y646N, Y646F or Y646X (Y646H, Y646S, or Y646C), A682G, and A692V of the EZH2 gene in DNA extracted from formalin fixed paraffin embedded (FFPE) human follicular lymphoma tumor tissue specimens. The cobas® EZH2 Mutation Test is intended for the identification of follicular lymphoma patients with an EZH2 mutation for treatment with TAZVERIK™ (tazemetostat), in accordance with the approved therapeutic product labeling.Specimens are processed using the cobas® DNA Sample Preparation Kit for manual sample preparation and the cobas z 480 analyzer for automated amplification and detection. en The cobas EZH2 Test is based on two major processes: (1) manual specimen preparation to obtain DNA from Formalin Fixed Paraffin Embedded Tissue (FFPET); and (2) PCR amplification and detection of target DNA using complementary primer pairs and oligonucleotide probes labeled with fluorescent dyes. Mutation detection is achieved through PCR analysis with the cobas z 480 analyzer. A mutant control and negative control are included in each run to confirm the validity of the run.Sample preparationFFPET specimens are processed and genomic DNA isolated using the cobas® DNA Sample Preparation Kit for manual specimen preparation based on nucleic acid binding to glass fibers. A deparaffinized 5-μm section of an FFPET specimen is lysed by incubation at an elevated temperature with a protease and chaotropic lysis/binding buffer that releases nucleic acids and protects the released genomic DNA from DNases. Subsequently, isopropanol is added to the lysis mixture that is then centrifuged through a column with a glass fiber filter insert. During centrifugation, the genomic DNA is bound to the surface of the glass fiber filter. Unbound substances, such as salts, proteins and other cellular impurities, are removed by centrifugation. The adsorbed nucleic acids are washed and then eluted with an aqueous solution. The amount of genomic DNA is determined by spectrophotometer and adjusted to a fixed concentration to be added to the amplification/detection mixture. The target DNA is then amplified and detected on the cobas z 480 analyzer using the amplification and detection reagents provided in the cobas EZH2 Test.PCR amplificationTarget selectionThe cobas EZH2 Test uses primers that define specific base-pair sequences for each of the targeted mutations with base-pair sequences ranging from 94 to 104 base pairs long in exons 16 and 18 and 100 base pairs for the internal control in exon 11. Amplification occurs only in the regions of the EZH2 gene between the primers; the entire EZH2 gene is not amplified.Target amplificationA derivative of Thermus species Z05-AS1 DNA polymerase is utilized for target amplification. First, the PCR mixture is heated to denature the genomic DNA and expose the primer target sequences. As the mixture cools, the upstream and downstream primers anneal to the target DNA sequences. The Z05-AS1 DNA polymerase, in the presence of divalent metal ion and excess dNTP, extends each annealed primer, thus synthesizing a second DNA strand. This completes the first cycle of PCR, yielding a double-stranded DNA copy which includes the targeted base-pair regions of the EZH2 gene. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of amplicon DNA.Automated real-time mutation detectionThe cobas EZH2 Test utilizes real-time PCR technology. Each target-specific, oligonucleotide probe in the reaction is labeled with a fluorescent dye that serves as a reporter, and with a quencher molecule that absorbs (quenches) fluorescent emissions from the reporter dye within an intact probe. During each cycle of amplification, probe complementary to the single-stranded DNA sequence in the amplicon binds and is subsequently cleaved by the 5’ to 3’ nuclease activity of the Z05-AS1 DNA Polymerase. Once the reporter dye is separated from the quencher by this nuclease activity, fluorescence of a characteristic wavelength can be measured when the reporter dye is excited by the appropriate spectrum of light. Four different reporter dyes are used to detect the EZH2 sequences targeted by the test. Amplification of the targeted EZH2 sequences are detected independently across three reactions by measuring fluorescence at the four characteristic wavelengths in dedicated optical channels.Selective amplificationSelective amplification of target nucleic acid from the sample is achieved in the cobas EZH2 Test by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP).6 The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine but not DNA containing thymidine. Deoxyuridine is not present in naturally occurring DNA but is always present in amplicon due to the use of dUTP in place of deoxythymidine triphosphate as one of the nucleotide triphosphates in the Master Mix reagents; therefore, only amplicon contains deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by AmpErase enzyme prior to amplification of the target DNA. The AmpErase enzyme, which is included in the Master Mix reagents, catalyzes the cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the C1-position. When heated in the first thermal cycling step at alkaline pH, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme is inactive at temperatures above 55°C, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon.6. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-8. PMID: 2227421. en

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